Yoshinori Minami

Nicotinamide phosphoribosyltransferase: a potent therapeutic target in non-small cell lung cancer with epidermal growth factor receptor-gene mutation.

Background: Non-small cell lung cancer (NSCLC) often has an epidermal growth factor receptor (EGFR) gene mutation. Growth of EGFR-gene-mutated NSCLC depends predominantly on EGFR signaling and requires a large amount of intracellular ATP to activate EGFR signal transduction. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis, and it regulates intracellular ATP levels in mammalian cells. The effect of NAMPT inhibition on NSCLC has not been completely understood. Methods: We aimed to clarify the hypothesis that NAMPT inhibition suppresses growth of EGFR-gene-mutated NSCLC through reduction of intracellular ATP levels, using NAMPT-siRNA transfection and NAMPT inhibitor FK866. We used four lung adenocarcinoma cell lines, including H358 (Wild type EGFR), LC2 (EGFRL858R), PC9 (EGFRDel E746-A750), and H1975 (EGFRL858R+T790M), and evaluated the effect of FK866 on these cells and its mechanisms, using cell proliferation, Western blot, ATP, and apoptosis assay. Results: We found that (1) H358, LC2, and H1975 cell lines highly expressed NAMPT-mRNA; (2) NAMPT-specific siRNA and FK866 suppressed proliferation of these NSCLCs; (3) FK866 reduced intracellular ATP levels in H1975 cells; (4) FK866 dephosphorylated EGFR signal proteins, including EGFR, Akt, Map kinase kinase 1/2, and extracellular signal-regulated kinase 1/2 (ERK 1/2); (5) FK866 induced apoptosis of H1975 cells; and (6) FK866 suppressed growth of H1975 xenograft tumors and attenuated expression of phospho-ERK 1/2 in the tumors in a tumor-bearing mouse model. Conclusion: These findings indicate that NAMPT is a potent therapeutic target in the treatment of EGFR-gene-mutated NSCLC.

Expression of Notch1 and Numb in small cell lung cancer

Notch signaling in tumorigenesis functions as an oncogene or tumor suppressor according to the type of malignancy. Numb represses intracellular Notch signaling. Previous studies have demonstrated that Notch signaling suppresses the proliferation of small cell lung cancer (SCLC) cell lines. However, in SCLC, the association between Notch1 and Numb expression and clinicopathological factors or prognosis has remained unclear. In this study, we evaluated the expression of Notch1 and Numb in SCLC. We immunohistochemically assessed 125 SCLCs that were surgically resected at 16 institutions participating in either the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) or the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) between 2003 and 2013. Correlations between Notch1 or Numb expression and various clinicopathological features were evaluated. Notch1 expression was associated with ECOG performance status. Numb expression was associated with age, sex, and pathological histology (SCLC or Combined SCLC). Analysis of cellular biological expression did not demonstrate a significant correlation between the expression of Notch1 and of Numb. Multivariate Cox regression analysis showed that high Notch1 expression was an independent favorable prognostic factor for SCLC(hazard ratio = 0.503, P = 0.023). High Notch1 expression, but not Numb expression, is associated with favorable prognosis in SCLC.

Prognostic impact of clinical variables on surgically resected small-cell lung cancer: Results of a retrospective multicenter analysis (FIGHT002A and HOT1301A)

OBJECTIVES Several American and Japanese guidelines recommend surgery for patients with c-stage I small-cell lung cancer (SCLC), whereas the European Society of Medical Oncology (ESMO) guidelines recommend surgery for patients with not only c-stage I but also c-stage II (T2N1) SCLC. In addition, previous studies identified various factors other than clinical stage that are related to survival in these patients. Thus, further validation and examination of the association of clinical stage and other clinical variables with survival are required for establishing practical management of early-stage SCLC. PATIENTS AND METHODS We reviewed the clinical courses of 156 SCLC patients who had undergone surgery at 17 institutions between January 2003 and January 2013. RESULTS Clinical stages (tumor-node-metastasis [TNM] version 7) of the 156 patients were 98 cases in IA, 14 in IB, 16 in IIA, 7 in IIB, 18 in IIIA, and 3 in IIIB. Median overall survival (OS) was 33.3 months (95% confidence interval: 20.9-45.8). Multivariate analysis revealed that OS was longer in patients either at c-stage II and under, with a maximum tumor diameter of <20mm, with preoperative diagnosis, without a history or presence of other types of cancer, or who underwent prophylactic cranial irradiation (PCI). CONCLUSION These results indicate that a history or presence of other types of cancer might be a major decisive factor for surgery. Patients with c-stages I and II (c-T2N1) can be considered for surgery, and PCI may be useful in patients undergoing surgery in a practical setting, partly supporting the ESMO guidelines.(1).

Prostaglandin I2 analog suppresses lung metastasis by recruiting pericytes in tumor angiogenesis.

Prostaglandin I2 (PGI2) agonist has been reported to reduce tumor metastasis by modifying tumor angiogenesis; however, the mechanisms of how PGI2 affects the endothelial cells or pericytes in tumor vessel maturation are still unclear. The purpose of this study was to clarify the effects of PGI2 on tumor metastasis in a mouse lung metastasis model using Lewis lung carcinoma (LLC) cells. The mice were treated continuously with beraprost sodium (BPS), a PGI2 analog, for 3 weeks and then examined for lung metastases. The number and size of lung metastases were decreased significantly by BPS treatment. In addition, scanning electron microscopy and immunohistochemistry revealed that BPS increased the number of tumor‑associated pericytes and improved intratumor hypoxia. Collectively, this study suggests that BPS attenuated vascular functional maturation in metastatic tumors.

Immunohistochemical profiling of receptor tyrosine kinases, MED12, and TGF-βRII of surgically resected small cell lung cancer, and the potential of c-kit as a prognostic marker

The limited number of available treatments for patients with small-cell lung cancer (SCLC) has prompted us to further investigate the biology of SCLC by molecular profiling. We collected formalin-fixed paraffin-embedded tumor samples from 127 patients with SCLC, who had undergone surgery at 16 institutions between January 2003 and January 2013, and analyzed the association between disease-specific survival and protein expression of c-kit, c-Met, epidermal growth factor receptor, human EGFR-related 2, vascular endothelial growth factor receptor II, anaplastic lymphoma kinase, mediator complex subunit 12 (MED12), and transforming growth factor beta receptor II (TGF-βRII) by immunohistochemistry (IHC). Of the 125 evaluable samples, all tumors expressed MED12, and 123 samples (98.4%) expressed TGF-βRII. MED12 was highly expressed in the nucleus in 92% of the positive samples while TGF-βRII was highly expressed in the cytoplasm in 55% of the positive samples. High c-kit expression was an independent favorable prognostic marker confirmed by multivariate analysis (hazard ratio: 0.543, 95% confidence interval: 0.310–0.953, p = 0.033). Both the relapse free-survival and overall survival of patients who underwent adjuvant chemotherapy were statistically longer in those with high c-kit expression (n = 38) than those with intermediate, low, or no c-kit expression (n = 19) (not reached vs 11.6 months, p = 0.021; not reached vs 25.9 months, p = 0.028). IHC for c-kit may offer a prognostic marker for early-stage SCLC, and the results for MED12 and TGF-βRII may suggest the biological characteristics of SCLC. Further investigation of the roles of their related molecules in early stage SCLC is required.

Histamine H1 antagonist levocetirizine as a potential cause of lung injury

Histamine H1 antagonists rarely cause drug‐induced lung injury (DLI). A woman in her 60s, who had been taking antihistaminic levocetirizine for 2 months, presented with progressive cough and shortness of breath. A chest radiograph showed patchy infiltrations on both lower lung fields. Chest computed tomography findings were consistent with non‐specific interstitial pneumonia. Serum markers associated with interstitial pneumonias were elevated. Room air arterial blood gas analysis revealed hypoxemia. Restrictive ventilatory impairment was noted with reduced diffusing capacity. Transbronchial lung biopsy specimens demonstrated unclassifiable alveolitis. Steroid pulse therapy was introduced for respiratory distress, but the initial response to treatment was poor. A drug lymphocyte stimulation test was positive for levocetirizine. The interstitial pneumonia improved following withdrawal of levocetirizine. Her illness has not recurred under steroid therapy and the discontinuation of levocetirizine. Antihistaminics may have a potential risk of DLI.

Accessory Cells in Tumor Angiogenesis — Tumor-Associated Pericytes

In contrast to the normal tissue vasculature, tumor vessels are structurally and functionally abnormal [1-3]. These abnormal tumor vessels are characterized by an irregular, disorganized, and tortuous architecture with a highly dysfunctional and leaky endothelial cell (EC) layer [1, 3]. ECs are often loosely connected with each other and are covered by fewer and abnormal mural pericytes (PCs) [2-4].

Neuromyelitis optica spectrum disorder secondary to treatment with anti-PD-1 antibody nivolumab: the first report

BackgroundImmune checkpoint blockade is developed as standard treatment for non-small cell lung cancer. However immune-related adverse events (irAE) have still unknown complications. Here, we report a patient with lung squamous cell carcinoma who developed neuromyelitis optica spectrum disorder with nivolumab.Case presentationA 75-year-old Japanese man with lung squamous cell carcinoma was administered nivolumab as second-line treatment. Two months after treatment with nivolumab, he presented acute paralysis in the bilateral lower limbs, sensory loss. Spinal magnetic resonance imaging showed T2 hyperintense lesions between C5-6 and Th12-L1. He was diagnosed with neuromyelitis optica spectrum disorder (NMOSD) by anti-aquaporin-4 antibody-positive in the serum and other examinations. After treatment, steroid reactivity was poor.ConclusionThis is the first patient who developed anti-AQP4 antibody-positive NMOSD as a nivolumab-induced irAE. Clinicians should be aware of this kind of potential neurological complication by using immune check point inhibitor and start the treatment of this irAE as soon as possible.

Abstract 4656: The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Background]In recent years, tumor genotyping is critical for making decisions of non-small cell lung cancer (NSCLC) treatment. However genetic rearrangement of ALK, RET, ROS1 and NTRK1 were identified in the subset of NSCLC patients, these clinical characteristics were still remained to be clear. Immunohistochemistry (IHC) is widely used as a simple and quick method for screening for protein expression of these oncogenes. Fluorescence in situ hybridization (FISH) or RT-PCR are performed consecutively to confirm the genotyping. However it is often taking time and expensive and it is needed to consolidate novel substitute methods. [Purpose]We performed the in situ hybridization (ISH) analysis to detect cellular mRNA in formalin-fixed paraffin-embedded (FFPE) tissue samples of lung cancer. We also conducted IHC with the use of ALK, RET, ROS1 and NTRK1 specific antibodies and investigate a comparative study to assess the usefulness of mRNA-ISH to detect expression of these mRNA. [Materials and Methods]We made tissue micro array (TMA) slides using 40 FFPE samples of resected lung adenocarcinoma with known epidermal growth factor receptor (EGFR) mutation status by PCR (PNA-LNA PCR clamp method). The mRNA in situ hybridization was employed using RNA scope R 2.0 Regant Kit with targeting probes. IHC staining was conducted by antibodies using 5A4 and D5F3 for ALK, RET01 for RET, D4D6 for ROS1, and 14G6 for NTRK1. [Results]All of the IHC stained cases were clearly detected by mRNA-ISH respectively. There were some cases which were only visualized by ISH, each case was partially positive and not totally of the tumor. ALK: 5% of cases (2 out of 40) were detected in mRNA-ISH and were strongly stained with both 5A4 and D5F3. RET: 20% of cases (8/40) were detected in mRNA-ISH. Two cases (5% of total) had positive RET01 staining, while 6 cases had negative staining. (3 had no other genetic alterations, 2 had EGFR-L858R confirmed by PCR, and 1 had ALK by IHC and FISH.) ROS1: 20% (8/40) were detected in mRNA-ISH. Three cases (7.5% of total) presented positive D4D6 staining, while 5 had negative staining. (3 had no other alterations, 1 had EGFR-E746-A750 and 1 had EGFR-L858R by PCR.) NTRK1: 17.5% of cases (7/40) were detected in mRNA-ISH. 1 case (2.5% of total) presented positive 14G6 staining, while 6 cases had negative staining. (2 had no other alterations, 2 had EGFR-L858R, 1 had EGFR-E746-A750, and 1 had ALK.) [Conclusion] The novel mRNA in situ hybridization method combined with traditional IHC could improve detection rate of ALK, RET, ROS1 and NTRK1 gene alterations in NSCLC FFPE samples. Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinori Minami, Yoshinobu Ohsaki. The novel mRNA in situ hybridization method for the detection of ALK, RET, ROS1 and NTRK1 mRNA in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2014-4656

Abstract 3720: Intrinsic and acquired resistance mechanisms of alectinib in ALK rearranged cells

Background However potency of Alectinib, a selective ALK inhibitor, for ALK rearranged NSCLC and for that with secondary mutation have been reported, intrinsic and acquired resistance are likely to emerge. Recent study showed tumor microenvironment triggered resistance to Crizotinib and TAE684. In this study, we examine whether emerging of intrinsic and acquired resistance are affected by tumor microenvironment in H3122, EML4-ALK positive lung cancer cell line, and in Alectinib-resistant clone of H3122 (AFR) which is generated in vitro. Elucidating intrinsic resistant mechanisms and preceding development of acquired resistance to understand its mechanisms will provide beneficial information for the development of novel therapeutic strategies in clinical practice. Methods We examined the effect of EGF produced by endothelial cells on Alectinib sensitivity of H3122 cell line, and EGF and HGF produced by fibroblasts on Alectinib sensitivity of H3122AFR by clonogenic assay. Downstream pathways of ALK in both cells were analyzed by western blotting. Combination treatments of Alectinib and other TKI were tested both in vitro and in vivo. Results H3122 cells which had been cultured with EGF acquired resistance to Alectinib treatment. Combination treatment of Alectinib and Erlotinib overcome EGF induced resistance and showed down-regulation in phosphorylation of AKT and ERK1/2. H3122AFR cells were also reduced their sensitivity to Alectinib at 100 uM or higher concentration when the media contained EGF or HGF. Erlotinib or Crizotinib combined with Alectinib canceled the EGF-induced or HGF-induced resistance by down-regulating phosphorylation of AKT and ERK1/2. In vivo, tumor xenografts of nude mice which had been orally administered Alectinib and Erlotinib showed tumor regression in both H3122 and H3122AFR, and those received Alectinib and Crizotinib showed regression in H3122AFR. Conclusion In our study, we showed ligands produced by tumor microenvironment, such as EGF and HGF, contributed the emerging of intrinsic resistance and acquired resistance by activating survival pathway. Further, the efficacy of combination treatment of Alectinib with Erlotinib or that with Crizotinib was shown. Collectively, Alectinib combined with EGFR-TKI would be an effective strategy for Alectinib-resistant lung cancer, and Alectinib with crizotinib is another considerable strategy. Citation Format: Yukiko Hibino, Sasaki Takaaki, Yoshinori Minami, Pasi A. Janne, Yoshinobu Ohsaki. Intrinsic and acquired resistance mechanisms of alectinib in ALK rearranged cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3720. doi:10.1158/1538-7445.AM2014-3720